The successful use of tissue- or tumor-selective promoters in targeted gene therapy for cancer depends on high and selective activity. Tg is a thyroid-specific protein that is expressed in the normal thyroid and a majority of thyroid tumors. In the present study, we show, using a luciferase reporter assay, that a construct containing the putative Tg promoter and enhancer is active in 4 thyroid carcinoma cell lines (including 2 anaplastic thyroid carcinoma cell lines) and not in 5 cancer cell lines arising from nonthyroid tissues. Furthermore, both the activity and the specificity of this construct were increased by pretreatment with 8-Br-cAMP and the histone deacetylase inhibitor depsipeptide (FR901228). Expression of thymidine kinase in thyroid cancer cells infected with a recombinant adenovirus (Ad) carrying a Tg enhancer/promoter-thymidine kinase expression cassette (AdTg enhancer/promoter-TK) correlated with the level of Tg enhancer/promoter activity in these cells. Under similar conditions, TK expression was not observed in cancer cell lines arising from nonthyroid tissues. Cells infected with AdTg enhancer/promoter-TK demonstrated preferential GCV sensitivity, with up to a 100,000-fold increase in GCV sensitivity in thyroid cancer cell lines compared to cancer cell lines of nonthyroid origin. The construct described herein can be used to selectively target thyroid cancer cells, and its expression can be modulated to further increase its specificity and selectivity, especially in anaplastic thyroid carcinoma cells, using 8-Br-cAMP and depsipeptide. Published 2002 Wiley-Liss, Inc.